The utilization of organized supramolecular assemblies to exploit the synergistic interactions afforded by close proximity, both for enzymatic synthesis and for the degradation of recalcitrant substrates, is an emerging theme in cellular biology. Anaerobic bacteria harness a multiprotein complex, termed the ''cellulosome,'' for efficient degradation of the plant cell wall. This megadalton catalytic machine organizes an enzymatic consortium on a multifaceted molecular scaffold whose ''cohesin'' domains interact with corresponding ''dockerin'' domains of the enzymes. Here we report the structure of the cohesin-dockerin complex from Clostridium thermocellum at 2.2-Å resolution. The data show that the ␤-sheet cohesin domain interacts predominantly with one of the helices of the dockerin. Whereas the structure of the cohesin remains essentially unchanged, the loop-helix-helix-loop-helix motif of the dockerin undergoes conformational change and ordering compared with its solution structure, although the classical 12-residue EF-hand coordination to two calcium ions is maintained. Significantly, internal sequence duplication within the dockerin is manifested in near-perfect internal twofold symmetry, suggesting that both ''halves'' of the dockerin may interact with cohesins in a similar manner, thus providing a higher level of structure to the cellulosome and possibly explaining the presence of ''polycellulosomes.'' The structure provides an explanation for the lack of cross-species recognition between cohesin-dockerin pairs and thus provides a blueprint for the rational design, construction, and exploitation of these catalytic assemblies.

The assembly of proteins that display complementary activities into macromolecular complexes is critical to cellular function. One such enzyme complex, of environmental significance, is the plant cell wall degrading apparatus of anaerobic bacteria, termed the cellulosome. The complex assembles through the interaction of enzyme-derived ''type I dockerin'' modules with the multiple ''cohesin'' modules of the scaffolding protein. Clostridium thermocellum type I dockerin modules contain a duplicated 22-residue sequence that comprises helix-1 and helix-3, respectively. The crystal structure of a C. thermocellum type I cohesin-dockerin complex showed that cohesin recognition was predominantly through helix-3 of the dockerin. The sequence duplication is reflected in near-perfect 2-fold structural symmetry, suggesting that both repeats could interact with cohesins by a common mechanism in wild-type (WT) proteins. Here, a helix-3 disrupted mutant dockerin is used to visualize the reverse binding in which the dockerin mutant is indeed rotated 180 o relative to the WT dockerin such that helix-1 now dominates recognition of its protein partner. The dual binding mode is predicted to impart significant plasticity into the orientation of the catalytic subunits within this supramolecular assembly, which reflects the challenges presented by the degradation of a heterogeneous, recalcitrant, insoluble substrate by a tethered macromolecular complex.cellulosome structure ͉ cellulosome assembly ͉ Clostridium thermocellum ͉ cohesin-dockerin

The objective was to characterize the fatty acid (FA) composition of lamb meat with emphasis on biohydrogenation intermediates (BI) induced by dietary sunflower and linseed oil and to test if a synergistic effect on meat trans-11 18:1 and cis-9,trans-11 18:2 concentrations could be obtained with a blend of both oils. Thirty two lambs were assigned to four groups and fed for 6 weeks one of the following diets: pelleted dehydrated lucerne (Control); and Control supplemented with 7.4% of sunflower oil (SF), linseed oil (LS) or a blend of sunflower and linseed oils (2 : 1 vol/vol) (SFLS). Longissimus thoracis muscles were analyzed for FA. LS increased n-3 PUFA due to contribution of 18:3n-3 but not of very long n-3 PUFA. Total conjugated linoleic acids were similar in oil-supplemented lambs, but the cis-9,trans-11 18:2 was higher with SF than with LS. No synergistic effects on trans-11 18:1 or cis-9,trans-11 18:2 were observed when both oils were fed together. Oil supplementation increased the concentrations of most BI in meat. However, the BI patterns were different for LS and SF. Some FA were only found in lambs fed linseed oil, including the unusual cis-12,cis-15 18:2 which is proposed as a new intermediate of the 18:3n-3 biohydrogenation pathway.

No abstract

The plant cell wall degrading apparatus of anaerobic bacteria includes a large multienzyme complex termed the "cellulosome." The complex assembles through the interaction of enzyme-derived dockerin modules with the multiple cohesin modules of the noncatalytic scaffolding protein. Here we report the crystal structure of the Clostridium cellulolyticum cohesindockerin complex in two distinct orientations. The data show that the dockerin displays structural symmetry reflected by the presence of two essentially identical cohesin binding surfaces. In one binding mode, visualized through the A16S/L17T dockerin mutant, the C-terminal helix makes extensive interactions with its cohesin partner. In the other binding mode observed through the A47S/F48T dockerin variant, the dockerin is reoriented by 180°and interacts with the cohesin primarily through the N-terminal helix. Apolar interactions dominate cohesin-dockerin recognition that is centered around a hydrophobic pocket on the surface of the cohesin, formed by Leu-87 and Leu-89, which is occupied, in the two binding modes, by the dockerin residues Phe-19 and Leu-50, respectively. Despite the structural similarity between the C. cellulolyticum and Clostridium thermocellum cohesins and dockerins, there is no cross-specificity between the protein partners from the two organisms. The crystal structure of the C. cellulolyticum complex shows that organism-specific recognition between the protomers is dictated by apolar interactions primarily between only two residues, Leu-17 in the dockerin and the cohesin amino acid Ala-129. The biological significance of the plasticity in dockerin-cohesin recognition, observed here in C. cellulolyticum and reported previously in C. thermocellum, is discussed.

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The enzymatic degradation of plant cell wall xylan requires the concerted action of a diverse enzymatic syndicate. Among these enzymes are xylan esterases, which hydrolyze the O-acetyl substituents, primarily at the O-2 position of the xylan backbone. All acetylxylan esterase structures described previously display a ␣/␤ hydrolase fold with a "Ser-His-Asp" catalytic triad. Here we report the structures of two distinct acetylxylan esterases, those from Streptomyces lividans and Clostridium thermocellum, in native and complex forms, with x-ray data to between 1.6 and 1.0 Å resolution. We show, using a novel linked assay system with PNP-2-O-acetylxyloside and a ␤-xylosidase, that the enzymes are sugar-specific and metal ion-dependent and possess a single metal center with a chemical preference for Co 2؉ . Asp and His side chains complete the catalytic machinery. Different metal ion preferences for the two enzymes may reflect the surprising diversity with which the metal ion coordinates residues and ligands in the active center environment of the S. lividans and C. thermocellum enzymes. These "CE4" Many plant cell wall polysaccharides, including xylan, mannan, and pectin are present in acetylated forms. Acetylation not only modifies the physicochemical properties of polysaccharides, notably increasing the solubility for matrix applications, but also means they are less readily attacked by phytopathogen-derived cell wall-degrading endoglycosidases. To overcome the steric problems provided by acetyl substituents, plant cell wall degrading microorganisms have developed a host of acetyl esterases whose function is to deacetylate the polysaccharides prior to, or concomitant with, its complete hydrolysis by a consortium of exo-and endo-acting glycoside hydrolases. Such microbial esterases have, unsurprisingly, found widespread industrial application in both biomass conversion and for the chemoenzymatic synthesis of diverse esters (see for example Refs. 1 and 2 and reviewed in Ref.3).Xylan is a chemically and structurally complex plant cell wall polysaccharide, whose complete degradation requires the action of a dedicated enzymatic consortium, . Thus far, structures of ferulate and acetylxylan esterases have revealed a ␣/␤ hydrolase fold, and they display a Ser-His-Asp catalytic triad. Examples include the family CE1 (predominantly bacterial) ferulate esterases (8 -10), the currently unclassified fungal ferulate esterases (11-13) and the xylan and xylooligosaccharide esterases from families CE5 (14, 15), CE6 (putative esterases, PDB code 2APJ, Centre for Eukaryotic Structural Genomics), and CE7 (16). Enzymes in the largest sequence-based esterase family, CE4, do not, however, display the standard ␣/␤ hydrolase fold.The carbohydrate esterase family CE4 contains over 870 open reading frames.3 This family is notable, not merely for its size, but also as many CE4 members have been reported to be metal ion-dependent. Furthermore, family CE4 contains members with both classical de-Oacetylase activity, such as the acetylxylan est...

Enzymes that hydrolyze complex carbohydrates play important roles in numerous biological processes that result in the maintenance of marine and terrestrial life. These enzymes often contain noncatalytic carbohydrate binding modules (CBMs) that have important substrate-targeting functions. In general, there is a tight correlation between the ligands recognized by bacterial CBMs and the substrate specificity of the appended catalytic modules. Through high-resolution structural studies, we demonstrate that the architecture of the ligand binding sites of 4 distinct family 35 CBMs (CBM35s), appended to 3 plant cell wall hydrolases and the exo-␤-D-glucosaminidase CsxA, which contributes to the detoxification and metabolism of an antibacterial fungal polysaccharide, is highly conserved and imparts specificity for glucuronic acid and/or ⌬4,5-anhydrogalaturonic acid (⌬4,5-GalA). ⌬4,5-GalA is released from pectin by the action of pectate lyases and as such acts as a signature molecule for plant cell wall degradation. Thus, the CBM35s appended to the 3 plant cell wall hydrolases, rather than targeting the substrates of the cognate catalytic modules, direct their appended enzymes to regions of the plant that are being actively degraded. Significantly, the CBM35 component of CsxA anchors the enzyme to the bacterial cell wall via its capacity to bind uronic acid sugars. This latter observation reveals an unusual mechanism for bacterial cell wall enzyme attachment. This report shows that the biological role of CBM35s is not dictated solely by their carbohydrate specificities but also by the context of their target ligands.carbohydrate protein binding ͉ enzyme targeting ͉ plant cell wall degradation ͉ protein cell attachment ͉ X-ray crystallography